Plasma membrane plays an important role in cell life because all connections and interactions between cells and the environment must pass through the membrane. For instance, substances go into or out of a cell through the plasma membrane. Hormones and drugs act on cells via plasma membrane, and metabolism regulation, cell recognition and immunization, etc, all are related to and depend on the functions of plasma membrane.
Substances get into or out of a cell via plasma membrane. In live cells, plasma membrane is highly selective with regard to substance permeability. There are three major ways for substances to pass through plasma membrane: passive transportation, active transportation, and endocytosis and exocytosis.
Protein transportation in eukaryotes includes secretion, endocytosis, and exocytosis (Nature 1994 Nov. 3; 372(6501): 55-63).
Macromolecules, such as proteins, polynucleotides and polysaccharides can only pass through plasma membrane via endocytosis and exocytosis. The mechanism of transporting these macromolecules is different from that of transporting small molecules such as solutes and ions, and involves the orderly formation and fusion of small vesicles along the plasma membrane. Endocytosis and exocytosis are both active transportation, because they need energy supply. One important feature of endocytosis and exocytosis is that the macromolecules are enclosed in the vesicle rather than being mixed up with other macromolecules or organelles. This ensures that macromolecules outside or inside the membrane can be orderly transported. Rapid vesicular formation and fusion in large numbers are one of the basic traits of all eukaryotic cells.
Cells have complex internal membrane systems. A transporting vesicle obtains its contents from the donor organelle selectively, and fuses with the membrane in a highly selective manner as well. Accordingly, all vesicles must have surface markers which allow the recognition of the target membrane based on their sources and contents, and the target membrane must also have corresponding receptors. Though the mechanism of this recognition remains to be elucidated, a widely noticed theory is that the recognition involves proteins called SNAREs (“SNAP Receptors”). v-SNARE present on the vesicle membrane and t-SNARE present on target membrane have complementary structure and directs vesicular transportation. During the process, a transporting vesicle may examine many possible targets on the membranes before its v-SNARE finds the complementary t-SNARE. According to this theory the recognition is regulated by members of an enzyme family called Rab protein. Rab proteins examine whether the pairing between v-SNARE and t-SNARE is correct. Rab protein binds to the surface of the coated vesicle while the donor membrane is budding to form the vesicles. When the vesicle meets the target membrane, v-SNARE couples with SNARE for a sufficient period of time to allow Rab protein to hydrolyze GTP, and to anchor the vesicle on the target membrane, and subsequent fusion.
The fusion of the inner membranes is catalyzed by a specific fusion protein, which can overcome the inherent energy barrier. Little was known about the mechanism, other than that the process needs ATP, GTP, acyl-coenzyme A and several other proteins, two of which are known: NSF (N-ethylmaleimide-sensitive fusion protein) and SNAPs (soluble NSF attachment proteins). SNAPs shuttle between the fusing membrane and cytoplasma, and bind to v-SNARE on the vesicle membrane and t-SNARE on the target membrane, initiating the assembly of the fusion machinery, which catalyzes the fusion of the lipid bilayers.
SNARE is a family of proteins. The basic functions of SNARE include: (1) it plays a significant regulatory role in the recognition between vesicular and target membranes; and (2) it catalyzes the fusion of lipid bilayers. Besides, because NSF greatly influences the secretory process of platelets and neurotransmitter releases, SNARE, as receptors of NSF, is also important in those processes. (Blood. 1999 94: 1313-8; J. Neurosci. 1998 18: 10241-9). Therefore SNARE plays important roles in the transportation of macromolecules such as proteins, transportation in a cell, the influence of hormone and drugs on cells, metabolism regulation, cell recognition and immunity etc.
The Golgi complex is known to be involved in exocytosis. Some secretory proteins exit the cell through the Golgi complex. The Golgi complex is also a main site of carbohydrate synthesis, where glycoproteins and polysaccharides are synthesized. Aminopolysaccharides are also sulfated in the Golgi complex.
The Golgi complex is also a place for sorting and delivering the products of ER. A large portion of carbohydrates produced in the Golgi complex are attached to proteins and fatty acids from ER as oligosaccharide side chains. Some oligosaccheride groups function as markers and direct proteins to lysosomes or other cellular compartments.
Two types of SNARE have been found in the Golgi complex. Their molecular weight is 28KD and 27KD, respectively, and in humans are located on chromosomes 17q11 and 17q21. They contain a coiled-coil functional domain in the center and an anchoring site on the carboxyl terminal (Science 1996, 272: 1161-3).
In Vitro tests indicate that SNARE protein plays an important role in the recognition and fusion of vesicles and target membrane during the cellular transportation between ER and Golgi complex. (Science 1996, 272: 1161-3; J. Cell Biol. 1996, 133: 507-16)
When a vesicle touches the target membrane, SNARE integrates with soluble SNAP and NSF and forms a 20s complex which promotes membrane fusion. NSF acts as ATPase, probably supplying energy to overcome the energy barrier (Mammalian Genome 7: 850-852, 1996). N-ethylmaleimide-sensitive factor (NSF) is an ATPase which is related to vesicular fusion in eukaryotes. NSF together with SNAP decompose cis-SNARE complex through hydrolyzing ATP, leading to the formation of trans-SNARE complex. Therefore SNARE is important in the forming of proteins in ER-Golgi complex and some relevant transporting processes (Mol Cell 199 Jul. 4(1): 97-107).
Gene mapping data place human GS27 near the gene which causes hereditary hypertension, and there are suggestions that the gene encoding GS27 may be helpful in the diagnoses and treatment of hereditary hypertension (Genomics 1999; 57(2): 285-8).
Through amino acid sequence comparison, the present inventors identified a new human SNARE protein 25 (hSNARE25). It is homologous to SNARE-29KD in rat Golgi complex (database accession # AF035823). hSNARE25 is believed to have some biological functions as SNARE-29KD.
As mentioned above hSNARE25 plays an important role in the regulation of cell division and embryo development. Moreover the regulation process is believed to involve many proteins, so there is a need to identify more proteins involved in these processes, especially to identify their amino acid sequences. The isolation of the gene which codes for SNARE25 protein also forms a foundation for identifying the protein's function, both under healthy and pathologic conditions, and for developing diagnostic and treatment methods.